MHC class II epitopes of 5T4 antigen

ABSTRACT

There is provided an MHC class II peptide epitope from 5T4 antigen. In particular, there is provided a peptide epitope of 5T4 which comprises one of the following: (i) the minimal epitope from the amino acid sequence shown as SEQ ID No. 2; (ii) the minimal epitope from the amino acid sequence shown as SEQ ID No. 3; (iii) a minimal epitope from the region 281-420 of 5T4. There is also provided a vaccine comprising such a peptide (or precursor thereof) and its use to treat and/or prevent a disease, in particular a cancerous disease.

FIELD OF THE INVENTION

The present invention relates to peptide epitopes of 5T4 antigen, andtheir use in immunotherapy.

BACKGROUND TO THE INVENTION

Prior to the identification of specific human tumour antigens, manyclinical trials were performed attempting to immunise cancer patientsagainst either whole cancer cells or subcellular fractions form cancercells. The identification of genes encoding tumour antigens, however,has made it possible to develop specific immunotherapies based onattacking tumour cells bearing the identified antigens. A variety ofclinical approaches utilising these genes or gene products are possibleas summarised in the following table.

Active immunotherapy (“Cancer vaccines”) 1. Immunisation with: i)purified antigen ii) immunodominant peptide (native or modified) iii)“naked” DNA encoding the antigen iv) recombinant viruses encoding theantigen v) antigen presenting cells pulsed with protein or peptide (ortransfected with genes encoding the antigen) 2. Use of cytokineadjuvants such as IL-2 and IL-12 administered systemically or encoded bythe immunising vector Passive immunotherapy (“Adoptiveimmunotherapy”) 1. Transfer of cells sensitized in vitro to the specificantigen (bulk or cloned populations) 2. Transduction of effector cells(or stem cells) with genes encoding T cell receptors that recognisesepcific antigens.

Immunisation with intact protein has the potential advantage ofsimultaneously immunising against both class I and class II epitopes butrequires extensive and time-consuming efforts to purify large amounts oftumour antigen. The identification of class I and class II peptideswithin a tumour antigen makes it possible to immunise with high levelsof pure synthetic peptide. The peptide approach also has the advantagethat one can choose between a class I and a class II type response (ormixture) by choosing which epitopes to use. Immunisation with peptidealso means that subdominant and/or cryptic epitopes can be chosen (asthe need for antigen processing may be bypassed or reduced to a“trimming” role) in order to stimulate a different subset of T cells.Also the peptide may be modified (for example at their HLA class I or IIanchor sites) to increase their immunogenicity.

In the past few years, much attention has been given to the role of CD8+T cells in tumour immunity. Tumour-specific CD8+ CTLs have been shown tobe capable of lysing tumour cells directly and eradicating tumour massesin vivo in animal models. However, CD4+ T cells are also thought to playa critical role (Wang and Rosenberg (1999) Immunological Reviews170:85-100) and it may be that optimal cancer vaccines require theparticipation of both. CD4+ and CD8+ T cells.

A number of oncofoetal or tumour-associated antigens (TAAs) have beenidentified and characterised in human and animal tumours. In general,TAAs are antigens expressed during foetal development which aredownregulated in adult cells, and are thus normally absent or presentonly at very low levels in adults. Tumour cells have been observed toresume expression of TAAs, and the application of TAAs for tumourdiagnosis, targeting and immunotherapy has therefore been suggested.

The TAA 5T4 (see WO 89/07947) has been previously characterised. It is a72 kDa glycoprotein expressed widely in carcinomas, but having a highlyrestricted expression pattern in normal adult tissues (see Table 1). Itappears to be strongly correlated to metastasis in colorectal andgastric cancer. The full nucleic acid sequence of human 5T4 is known(Myers et al., 1994 J Biol Chem 169: 9319-24).

TABLE 1 Distribution of Human 5T4 Tumour 5T4 Type (%) Frequency Breast84 Ovarian 71 Gastric 74 Colorectal 85(Starzynska et al., Eur J Gastroenterol Hepatol 1998 June; 10(6):479-84;Starzynska et al., Br J Cancer 1994 May; 69(5):899-902; Starzynska etal., Br J Cancer 1992 November; 66(5):867-9)

5T4 has been proposed as a marker, with possible mechanisticinvolvement, for tumour progression and metastasis potential (Carsberget al., (1996) Int J Cancer 1996 Sep. 27; 68(1):84-92). 5T4 has alsobeen proposed for use as an immunotherapeutic agent (see WO 00/29428).

SUMMARY OF THE INVENTION

The present inventors have identified a number of MHC class I and IIrestricted epitopes of 5T4. The identification of particular antigenicpeptides provides new opportunities for the development of diagnosticand therapeutic strategies against cancer.

Thus the first aspect of the present invention relates to MHC class IIepitopes of 5T4 antigen. The invention provides peptide epitopes of 5T4antigen which are capable of being presented in conjunction with an MHCclass II molecule such that they are specifically recognised by a Tcell.

In particular, the present invention provides a peptide epitope of 5T4which comprises one of the following:

-   -   (i) the amino acid sequence shown as SEQ ID No. 2;    -   (ii) the minimal epitope from the amino acid sequence shown as        SEQ ID No. 3;    -   (iii) a minimal epitope from the region 281-420 of 5T4.

Further aspects of the invention relate to:

-   -   a polyepitope string comprising such a peptide.    -   such a peptide epitope, or such a polyepitope string in        association with a cell penetrator. A such a peptide epitope, or        such a polyepitope string associated with a tetramer.    -   a nucleic acid sequence capable of encoding such a peptide        epitope or polyepitope string (and optionally an associated cell        penetrator).    -   a vector system capable of delivering such a nucleic acid        sequence to a cell.    -   a cell pulsed with such a peptide epitope (or a precursor        thereof).    -   a vaccine comprising such a peptide epitope, a polyepitope        string, nucleic acid sequence, vector system and/or cell.    -   the use of such a vaccine in the manufacture of a medicament for        use in the prevention and/or treatment of a disease.    -   a method for treating and/or preventing a disease in a subject        in need of same which comprises the step of administering an        effective amount of such a vaccine to the subject.    -   an agent capable of binding specifically to such a peptide        and/or nucleic acid sequence.    -   a method which comprises the step of detecting the presence of        such a peptide, nucleic acid or agent in a subject.    -   a T cell line or clone capable of specifically recognising such        a peptide epitope in conjunction with an MHC class II molecule.

Other aspects of the present invention are presented in the accompanyingclaims and in the following description and discussion. These aspectsare presented under separate section headings. However, it is to beunderstood that the teachings under each section heading are notnecessarily limited to that particular section heading.

DETAILED DESCRIPTION OF THE INVENTION

Epitopes

The present invention relates to peptide epitopes.

The term “peptide” is used in the normal sense to mean a series ofresidues, typically L-amino acids, connected one to the other typicallyby peptide bonds between the α-amino and carboxyl groups of adjacentamino acids. The term includes modified peptides and synthetic peptideanalogues.

A T cell epitope is a short peptide derivable from a protein antigen.Antigen presenting cells can internalise antigen and process it intoshort fragments which are capable of binding MHC molecules. Thespecificity of peptide binding to the MHC depends on specificinteractions between the peptide and the peptide-binding groove of theparticular MHC molecule.

Peptides which bind to MHC class I molecules (and are recognised by CD8+T cells) are usually between 6 and 12, more usually between 8 and 10amino acids in length. The amino-terminal amine group of the peptidemakes contact with an invariant site at one end of the peptide groove,and the carboxylate group at the carboxy terminus binds to an invariantsite at the other end of the groove. The peptide lies in an extendedconfirmation along the groove with further contacts between main-chainatoms and conserved amino acid side chains that line the groove.Variations in peptide length are accomodated by a kinking in the peptidebackbone, often at proline or glycine residues.

Peptides which bind to MHC class II molecules are usually at least 10amino acids, for example about 13-18 amino acids in length, and can bemuch longer. These peptides lie in an extended confirmation along theMHC II peptide-binding groove which is open at both ends. The peptide isheld in place mainly by main-chain atom contacts with conserved residuesthat line the peptide-binding groove.

The peptide of the present invention may be made using chemical methods(Peptide Chemistry, A practical Textbook. Mikos Bodansky,Springer-Verlag, Berlin.). For example, peptides can be synthesized bysolid phase techniques (Roberge J Y et al (1995) Science 269: 202-204),cleaved from the resin, and purified by preparative high performanceliquid chromatography (e.g., Creighton (1983) Proteins Structures AndMolecular Principles, WH Freeman and Co, New York N.Y.). Automatedsynthesis may be achieved, for example, using the ABI 43 1 A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer.

The peptide may alternatively be made by recombinant means, or bycleavage from a longer polypeptide. For example, the peptide may beobtained by cleavage from full-length 5T4. The composition of a peptidemay be confirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure).

The term “peptide epitope” encompasses modified peptides. For example5T4 peptides may be mutated, by amino acid insertion, deletion orsubstitution, so long as the MHC binding-specificity of the wild-type5T4 peptide is retained. In a preferred embodiment the modified epitopehas greater affinity for the peptide binding groove. Preferably thepeptide contains 5 or fewer mutations from the wild-type sequence, morepreferably 3 or fewer, most preferably 1 or 0 mutations.

Alternatively (or in addition) modifications may be made withoutchanging the amino acid sequence of the peptide. For example, D-aminoacids or other unnatural amino acids can be included, the normal amidebond can be replaced by ester or alkyl backbone bonds, N-or C-alkylsubstituents, side chain modifications, and constraints such asdisulphide bridges and side chain amide or ester linkages can beincluded. Such changes may result in greater in vivo stability of thepeptide, and a longer biological lifetime.

Modification of epitopes may be performed based on predictions for moreefficient T-cell induction derived using the program “Peptide BindingPredictions” devised by K. Parker (NIH) which may be found on the worldwide web at bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform (seealso Parker, K. C et al. 1994. J. Immunol. 152:163).

A “modified” 5T4 peptide epitope includes peptides which have been boundor otherwise associated to transporter peptides or adjuvants, in orderto increase their ability to elicit an immune response. For example,peptides may be fused to TAP independent transporter peptides forefficient transport to HLA and interaction with HLA molecules to enhanceCTL epitopes (for review see Yewdell et al., 1998 J Immunother21:127-31; Fu et al., (1998) J Virol 72:1469-81).

In a further embodiment, 5T4 or 5T4 peptides may be fused to hepatitis Bcore antigen to enhance T helper and antibody responses (Schodel et al.,1996 Intervirology 39:104-10).

To be an epitope, the peptide should be capable of binding to thepeptide-binding groove of a MHC class I or II molecule and be recognisedby a T cell.

Cell surface presentation of peptides derived from a given antigen isnot random and tends to be dominated by a small number of frequentlyoccurring epitopes. The dominance of a particular peptide will depend onmany factors, such as relative affinity for binding the MHC molecule,spatio-temporal point of generation within the APC and resistance todegradation.

The epitope hierarchy for an antigen is thought to change withprogression of an immune response. After a primary immune response tothe immunodominant peptides, epitope “spreading” may occur tosub-dominant determinants (Lehmann et al (1992) Nature 358:155-157).

For any given antigen, cryptic epitopes may also exist. Cryptic epitopesare those which can stimulate a T cell response when administered as apeptide but which fail to produce such a response when administered as awhole antigen. It may be that during processing of the antigen intopeptides in the APC the cryptic epitope is destroyed.

The peptide of the invention may be an immunodominant epitope, asub-dominant epitope or a cryptic epitope of 5T4.

Epitopes for an antigen may be identified by measuring the T cellresponse to overlapping peptides spanning a portion of the antigen (seebelow) when presented by APC. Such studies usually result in “nestedsets” of peptides, and the minimal epitope for a particular T cellline/clone can be assessed by measuring the response to truncatedpeptides.

The minimal epitope for an antigen may not be the best epitope forpractical purposes. It may well be that amino acids flanking the minimalepitope will be required for optimal binding to the MHC.

Identification of Epitopes

There are a number of methods known in the art to identify the T cellepitopes within a given antigen.

Naturally processed epitopes may be identified by massspectrophotometric analysis of peptides eluted from antigen-loaded APC.These are APC that have either been encouraged to take up antigen, orhave been forced to produce the protein intracellularly bytransformation with the appropriate gene. Typically APC are incubatedwith protein either in solution or suitably targeted to the APC cellsurface. After incubation at 37° C. the cells are lysed in detergent andthe class II protein purified by, for example, affinity chromatography.Treatment of the purified MHC with a suitable chemical medium (forexample, acid conditions) results in the elution of peptides from theMHC. This pool of peptides is separated and the profile compared withpeptide from control APC treated in the same way. The peaks unique tothe protein expressing/fed cells are analysed (for example by massspectrometry) and the peptide fragments identified. This procedureusually generates information about the range of peptides (usually foundin “nested sets”) generated from a particular antigen by antigenprocessing.

Another method for identifying epitopes is to screen a synthetic libraryof peptides which overlap and span the length of the antigen in an invitro assay. For example, peptides which are 15 amino acids in lengthand which overlap by 5 or 10 amino acids may be used. The peptides aretested in an antigen presentation system which comprises antigenpresenting cells and T cells. For example, the antigen presentationsystem may be a murine splenocyte preparation, a preparation of humancells from tonsil or PBMC Alternatively, the antigen presentation systemmay comprise a particular T cell line/clone and/or a particular antigenpresenting cell type.

T cell activation may be measured via T cell proliferation (for exampleusing ³H-thymidine incorporation) or cytokine production. Activation ofTH1-type CD4+ T cells can, for example be detected via IFNγ productionwhich may be detected by standard techniques, such as an ELISPOT assay.

Overlapping peptide studies usually indicate the area of the antigen inwhich an epitope is located. The minimal epitope for a particular T cellcan then be assessed by measuring the response to truncated peptides.For example if a response is obtained to the peptide comprising residues1-15 in the overlapping library, sets which are truncated at both ends(i.e. 1-14, 1-13, 1-12 etc. and 2-15, 3-15, 4-15 etc.) can be used toidentify the minimal epitope.

Polyepitope String

It has been found that it is particularly effective way to induce animmune response to an antigen is by the use of a polyepitope string,which contains a plurality of antigenic epitopes from one or moreantigens linked together. For example, for malaria, a polyepitope stringof mainly malaria (P. falciparum) CD8 T cell peptide epitopes has beendescribed which also expresses CD4 T cell epitopes from tetanus toxoidand from the 38 Kd mycobacterial antigen of various strains of M.tuberculosis and M. bovis.

The present invention also provides a polyepitope string comprising atleast one peptide according to the present invention. The string mayalso comprise another epitope derivable from the 5T4 antigen or anepitope from another antigen—such as another TAA—or combinationsthereof.

TAAs have been characterised either as membrane proteins or alteredcarbohydrate molecules of glycoproteins and glycolipids, however theirfunctions remain largely unknown. One TAA family, the transmembrane 4superfamily (TM4SF), usually has four well-conserved membrane-spanningregions, certain cysteine residues and short sequence motifs. There isevidence that TM4SF antigens exist in close association with otherimportant membrane receptors including CD4 and CD8 of T cells (Imai &Yoshie (1993) J. Immunol. 151, 6470-6481). It has also been suggestedthat TM4SF antigens may play a role in signal transduction which inturn, affects cell development, activation and motility. Examples ofTM4SF antigens include human melanoma-associated antigen ME491, humanand mouse leukocyte surface antigen CD37, and human lymphoblasticleukemia-associated TALLA-1 (Hotta, H. et al. (1988) Cancer Res. 48,2955-2962; Classon, B. J. et al. (1989) J. Exp. Med. 169: 1497-1502;Tomlinson, M. G. et al. (1996) Mol. Immun. 33: 867-872; Takagi, S. etal. (1995) Int. J. Cancer 61: 706-715).

Further examples of TAAs also include, but are not limited to, TAAs inthe following classes: cancer testis antigens (HOM-MEL-40),differentiation antigens (HOM-MEL-55), overexpressed gene products(HOM-MD-21), mutated gene products (NY-COL-2), splice variants(HOM-MD-397), gene amplification products (HOM-NSCLC-11) and cancerrelated auto-antigens (HOM-MEL-2.4) as reviewed in Cancer Vaccines andImmunotherapy (2000) Eds Stern, Beverley and Carroll, CambridgeUniversity Press, Cambridge. Further examples include, MART-1 (MelanomaAntigen Recognised by T cells-1) MAGE-A (MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A10, MAGE-A12), MAGE B(MAGE-B1-MAGE-B24), MAGE-C (MAGE-C1/CT7, CT10), GAGE (GAGE-1, GAGE-8,PAGE-1, PAGE-4, XAGE-1, XAGE-3), LAGE (LAGE-1a(1S), -1b(1L), NY-ESO-1),SSX (SSX1-SSX-5), BAGE, SCP-1, PRAME (MAPE), SART-1, SART-3, CTp11,TSP50, CT9/BRDT, gp100, MART-1, TRP-1, TRP-2, MELAN-A/MART-1,Carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), MUCIN(MUC-1) and Tyrosinase. TAAs are reviewed in Cancer Immunology (2001)Kluwer Academic Publishers, The Netherlands.

Cell Penetrators

The present invention also provides a peptide epitope, or a polyepitopestring in association with a cell penetrator.

Antigen presenting cells (such as dendritic cells) pulsed with peptideshave proven effective in enhancing antitumour immunity (Celluzzi et al(1996) J. Exp. Med. 183 283-287; Young et al (1996) J. Exp. Med. 1837-11). It has been shown that it is possible to prolong the presentationof a peptide by dendritic cells (and thus enhance antitumour immunity)by linking it to a cell penetrating peptide (CPP) (Wang and Wang (2002)Nature Biotechnology 20 149-154).

A cell penetrator may be any entity which enhances the intracellulardelivery of the peptide/polyepitope string to the antigen presentingcell. For example, the cell penetrator may be a lipid which, whenassociated with the peptide, enhances its capacity to cross the plasmamembrane. Alternatively, the cell penetrator may be a peptide. Severalcell penetrating peptide (CPPs) have been identified from proteins,including the Tat protein of HIV (Frankel and Pabo (1988) Cell 551189-1193), the VP22 protein of HSV (Elliott and O'Hare (1997) Cell 88223-233) and fibroblast growth factor (Lin et al (1995) J. Biol. Chem.270 14255-14258).

The term “associated with” is intended to include direct linkage, forexample by a covalent bond. Examples of covalent bonds for linking aminoacids include disulphide bridges and peptide bonds. In a preferredembodiment, the peptide/polyepitope string and a CPP are linked by apeptide bond to create a fusion protein.

The term also includes non-covalent linkage, such as association byelectrostatic bonding, hydrogen bonding and van der Waals forces. Thecell penetrator and peptide/polyepitope string may be associated withoutcovalent or non-covalent bonding. For example the cell penetrator may bea lipid which encapsulates the peptide/polyepitope string (e.g.a.liposome).

5T4

5T4 has been previously characterised, for example, in WO89/07947. Thesequence of human 5T4 which appears in GenBank at accession no. Z29083.The peptide may also be derived from a 5T4 antigen from a differentspecies, such as murine 5T4 (WO00/29428), canine 5T4 (WO01/36486) orfeline 5T4 (SEQ ID No 1 presented herein). The peptide may also bederived from a naturally occurring variant of 5T4 found with aparticular species, preferably a mammal. Such a variant may be encodedby a related gene of the same gene family, by an allelic variant of aparticular gene, or represent an alternative splicing variant of the 5T4gene.

A peptide derived from 5T4 from a different species or a splice variantmay have a different amino acid sequence from the analogous humanwild-type 5T4 peptide. However, as long as the peptide retains the samequalitative binding specificity as the human peptide (i.e. it binds inthe peptide binding groove of an MHC molecule of the same haplotype)then it is still an epitope in accordance with the present invention.

Nucleic Acid

The present invention also relates to a nucleic acid sequence capable ofencoding a peptide epitope or polyepitope string according to the firstaspect of the invention.

A “nucleic acid”, as referred to herein, may be DNA or RNA,naturally-occurring or synthetic, or any combination thereof. Nucleicacids according to the invention are limited only in that they serve thefunction of encoding a 5T4 peptide in such a way that it may betranslated by the machinery of the cells of a host organism. Thus,natural nucleic acids may be modified, for example to increase thestability thereof. DNA and/or RNA, but especially RNA, may be modifiedin order to improve nuclease resistance of the members. For example,known modifications for ribonucleotides include 2′-O-methyl, 2′-fluoro,2′-NH₂, and 2′-O-allyl. The modified nucleic acids according to theinvention may comprise chemical modifications which have been made inorder to increase the in vivo stability of the nucleic acid, enhance ormediate the delivery thereof, or reduce the clearance rate from thebody. Examples of such modifications include chemical substitutions atthe ribose and/or phosphate and/or base positions of a given RNAsequence. See, for example, WO 92/03568; U.S. Pat. No. 5,118,672; Hobbset al., (1973) Biochemistry 12:5138; Guschlbauer et al., (1977) NucleicAcids Res. 4:1933; Schibaharu et al., (1987) Nucleic Acids Res. 15:4403;Pieken et al., (1991) Science 253:314, each of which is specificallyincorporated herein by reference.

The present invention also encompasses nucleic acids which willhybridise to a nucleic acid sequence capable of encoding a peptideepitope or polyepitope string according to the first aspect of theinvention.

Stringency of hybridisation refers to conditions under which polynucleicacid hybrids are stable. Such conditions are evident to those ofordinary skill in the field. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (Tm) of thehybrid which decreases approximately 1 to 1.5° C. with every 1% decreasein sequence homology. In general, the stability of a hybrid is afunction of sodium ion concentration and temperature. Typically, thehybridisation reaction is performed under conditions of higherstringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permithybridisation of only those nucleic acid sequences that form stablehybrids in 1 M Na+ at 65-68° C. High stringency conditions can beprovided, for example, by hybridisation in an aqueous solutioncontaining 6×SSC, 5×Denhardt's, 1% SDS (sodium dodecyl sulphate), 0.1Na+ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as nonspecific competitor. Following hybridisation, high stringency washingmay be done in several steps, with a final wash (about 30 min) at thehybridisation temperature in 0.2-0.1×SSC, 0.1% SDS.

Moderate stringency refers to conditions equivalent to hybridisation inthe above described solution but at about 60-62° C. In that case thefinal wash is performed at the hybridisation temperature in 1×SSC, 0.1%SDS.

Low stringency refers to conditions equivalent to hybridisation in theabove described solution at about 50-52° C. In that case, the final washis performed at the hybridisation temperature in 2×SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicatedusing a variety of buffers, e.g. formamide-based buffers, andtemperatures. Denhardt's solution and SSC are well known to those ofskill in the art as are other suitable hybridisation buffers (see, e.g.Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds.(1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.).Optimal hybridisation conditions have to be determined empirically, asthe length and the GC content of the probe also play a role.

Given the guidance provided herein, the nucleic acids of the inventionare obtainable according to methods well known in the art. For example,a DNA of the invention is obtainable by chemical synthesis, usingpolymerase chain reaction (PCR) or direct cleavage from a longerpolynucleotide, such as the entire 5T4 coding sequence or a fragmentthereof.

Chemical methods for synthesis of a nucleic acid of interest are knownin the art and include triester, phosphite, phosphoramidite andH-phosphonate methods, PCR and other autoprimer methods as well asoligonucleotide synthesis on solid supports. These methods may be usedif the entire nucleic acid sequence of the nucleic acid is known, or thesequence of the nucleic acid complementary to the coding strand isavailable. Alternatively, if the target amino acid sequence is known,one may infer potential nucleic acid sequences using known and preferredcoding residues for each amino acid residue.

It is envisaged that the nucleic acid of the invention can be modifiedby nucleotide substitution, nucleotide deletion, nucleotide insertion orinversion of a nucleotide stretch, and any combination thereof. Suchmutants can be used e.g. to produce a 5T4 peptide that has an amino acidsequence differing from the wild-type 5T4 epitope. Such a peptide isstill a peptide in accordance with the present invention if it retainsthe capacity to act as a T cell epitope. Mutagenesis may bepredetermined (site-specific) or random. A mutation which is not asilent mutation should not place sequences out of reading frames andpreferably will not create complementary regions that could hybridise toproduce secondary mRNA structure such as loops or hairpins.

Variants/Fragments/Homologues/Derivatives

The present invention encompasses the use of nucleotide and amino acidsequences and variants, homologues, derivatives and fragments thereof.

The term “variant” is used to mean a naturally occurring polypeptide ornucleotide sequence which differs from a wild-type sequence.

The term “fragment” indicates that a polypeptide or nucleotide sequencecomprises a fraction of a subject sequence. Preferably the sequencecomprises at least 50%, more preferably at least 65%, more preferably atleast 80%, more preferably at least 90%, most preferably at least 90% ofthe subject sequence. If the fragment is a fragment of an amino acidthen preferably the fragments are 6-12 amino acids in length.

The term “homologue” means an entity having a certain homology with thesubject amino acid sequences and the subject nucleotide sequences. Here,the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence, which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same activity as the subjectamino acid sequence. Although homology can also be considered in termsof similarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

In the present context, a homologous sequence is taken to include anucleotide sequence, which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical to the subject sequence.Typically, the homologues will comprise the same activity as the subjectsequence. Although homology can also be considered in terms ofsimilarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

Homology comparisons may be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example, when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However, for some applications, it is preferred to usethe GCG Bestfit program. A new tool, called BLAST 2 Sequences is alsoavailable for comparing protein and nucleotide sequence (see FEMSMicrobiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1):187-8).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix—such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues, which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example, according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) may occur i.e. like-for-like substitution—such as basic forbasic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids—such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids—such astrifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*,p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-aminocaproic acid^(#), 7-amino heptanoic acid*, L-methionine sulfone^(#*),L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*,L-hydroxyproline^(#), L-thioproline*, methyl derivatives ofphenylalanine (Phe)—such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe(4-amino)^(#), L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionicacid^(#) and L-Phe (4-benzyl)*. The notation * has been utilised for thepurpose of the discussion above (relating to homologous ornon-homologous substitution), to indicate the hydrophobic nature of thederivative whereas # has been utilised to indicate the hydrophilicnature of the derivative, #* indicates amphipathic characteristics.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups—such as methyl, ethyl or propyl groups—inaddition to amino acid spacers—such as glycine or β-alanine residues. Afurther form of variation involves the presence of one or more aminoacid residues in peptoid form will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example, Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences may be modified by any methodavailable in the art. Such modifications may be carried out to enhancethe in vivo activity or life span of nucleotide sequences useful in thepresent invention.

Vector System

The nucleic acid sequence of the present invention may be delivered to acell by way of a vector system.

As used herein, a “vector” may be any agent capable of delivering ormaintaining nucleic acid in a host cell, and includes viral vectors,plasmids, naked nucleic acids, nucleic acids complexed with polypeptideor other molecules and nucleic acids immobilised onto solid phaseparticles. Such vectors are described in detail below. It will beunderstood that the present invention, in its broadest form, is notlimited to any specific vector for delivery of the 5T4 peptide-encodingnucleic acid.

The vector may be a prokaryotic or eukaryotic vector.

Nucleic acids encoding 5T4 epitopes and polyepitope strings inaccordance with the present invention can be delivered by viral ornon-viral techniques.

Non-viral delivery systems include but are not limited to DNAtransfection methods. Here, transfection includes a process using anon-viral vector to deliver a 5T4 gene to a target mammalian cell.

Typical transfection methods include electroporation, nucleic acidbiolistics, lipid-mediated transfection, compacted nucleic acid-mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationicagent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology1996 14; 556), multivalent cations such as spermine, cationic lipids orpolylysine, 1,2,-bis (oleoyloxy)-3-(trimethylammonio) propane(DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 NatureBiotechnology 16: 421) and combinations thereof.

Non-viral delivery systems may also include, but are not limited to,bacterial delivery systems. The use of bacteria as anticancer agents andas delivery agents for anticancer drugs has been reviewed in Expert OpinBiol Ther 2001 March; 1(2):291-300.

Suitable bacteria include, but are not limited to, bacterial pathogensand non-pathogenic commensal bacteria. By way of example, suitablegenera may be selected from Salmonella, Mycobacterium, Yersinia,Shigella, Listeria and Brucella. Recent advances in the pathogenesis andmolecular biology of these bacteria have allowed the rationaldevelopment of new and improved bacterial carriers and more effectivegene expression systems. These advances have improved the performanceand versatility of these delivery systems.

The bacteria may be invasive intracellular bacteria that are able totransfer eukaryotic expression plasmids into mammalian host cells invitro and in vivo. Plasmid transfer may take place when the recombinantbacterium dies within the host cell, either due to metabolic attenuationor induction of autolysis. Alternatively, antibiotics may be used andspontaneous transfer has also been observed, indicating that thisphenomenon might also occur under physiological conditions. Plasmidtransfer has been reported for Shigella flexneri, Salmonellatyphimurium, S. typhi, Listeria monocytogenes and recombinantEscherichia coli, but other invasive bacteria may also be used.

Bacteria may be used for DNA vaccine delivery. Such bacteria may enterthe host cell cytosol after phagocytosis, for example, Shigella andListeria, or they remain in the phagosomal compartment—such asSalmonella. Both intracellular localisations may be suitable forsuccessful delivery of DNA vaccine vectors.

The bacterial delivery systems may utilise Mycobacterium in the form ofnon pathogenic Mycobacterium strains, genetic transfer systems in theform of cloning and expression vectors, and related technologies toprovide products containing, for example, non toxic immuno-regulatingMycobacterium adjuvants, non toxic immuno-stimulating exogenous antigensspecific for a variety of diseases, and non toxic amounts of cytokinesthat boost the TH-1 pathway (Tunis Med 2001 February; 79(2):65-81).

Salmonella strains—such as attenuated strains—which comprise definedgene deletions, may be used as suitable delivery systems—such as thedelivery of antigens. A number of strategies for delivery by thesestrains have been attempted, ranging from plasmid-based to chromosomalintegration systems. By way of example, Rosenkranz et al. Vaccine 2003,21(7-8), 798-801 describe eukaryotic expression plasmids encodingcytokines, and assessed their capacity to modulate immune responses indifferent experimental models. Plasmids encoding mouse IL-4 and IL-18under cytomegalovirus promoter were constructed and transformed intolive attenuated Salmonella enterica serovar Typhi strain CVD 908-htrA,and Salmonella enterica serovar Typhimurium strain SL3261.

The use of attenuated Salmonella typhimurium as a potential genedelivery vector has been reviewed in Anticancer Res 2002, 22(6A):3261-6.

Brucella abortus may also be used as a suitable delivery system asdescribed by Vemulapalli et al. Infect Immun (2000) 68(6):3290-6.Brucella abortus strain RB51 is a stable, rough, attenuated mutantwidely used as a live vaccine for bovine brucellosis. This strain may beused as a delivery vector, for example, in the delivery of protectiveantigens of other intracellular pathogens to which the induction of astrong Th1 type of immune response is needed for effective protection.

Boyd et al. Eur J Cell Biol (2000) 79 (10) 659-71 describe the use ofYersinia enterocolitica for the delivery of proteins into a wide rangeof cell types. Y. enterocolitica translocates virulence proteins, calledYop effectors, into the cytosol of eukaryotic cells. No limit to therange of eukaryotic cells into which Y. enterocolitica can translocateYops was reported. The Yop effectors YopE, YopH and YopT were eachcytotoxic for the adherent cell types tested, showing that not only isY. enterocolitica not selective in its translocation of particular Yopeffectors into each cell type, but also that the action of these Yopeffectors is not cell type specific. To use the Yersinia translocationsystem for broad applications, a Y. enterocolitica translocation strainand vector for the delivery of heterologous proteins into eukaryoticcells was constructed. This strain and vector combination lacks thetranslocated Yop effectors and allows delivery into eukaryotic cells ofheterologous proteins fused to the minimal N-terminalsecretion/translocation signal of YopE.

U.S. Pat. No. 5,965,381 describes a recombinant Yersinia for thedelivery of proteins into eukaryotic cells. Such Yersinia are deficientin the production of functional effector proteins, but are endowed witha functional secretion and translocation system.

Cell adhesion molecules are a large group of molecules involved in avariety of cell-to-cell and cell-to-extra-cellular matrix (ECM)interactions and are exploited by a number of pathogenic micro-organismsas receptors for cell entry. These molecules may be used for thetargeting and uptake of both gene and drug delivery systems. Celladhesion molecules and their use in gene transfer has been reviewed inAdv Drug Deliv Rev 2000 Nov. 15; 44(2-3):135-52.

The gene gun delivery system may also be used for the delivery of DNA,which is a highly reliable method compared to intramuscular inoculation(Jpn J Pharmacol 2000 July; 83(3):167-74).

Viral delivery systems include but are not limited to adenovirusvectors, adeno-associated viral (AAV) vectors, herpes viral vectors,retroviral vectors, lentiviral vectors or baculoviral vectors,Venezuelan equine encephalitis virus (VEE), poxviruses such as:canarypox virus (Taylor et al 1995 Vaccine 13:539-549), entomopox virus(Li Y et al 1998 XII^(th) International Poxvirus Symposium p144.Abstract), penguine pox (Standard et al. J Gen Virol. 1998 79:1637-46)alphavirus, and alphavirus based DNA vectors.

Examples of retroviruses include but are not limited to: murineleukaemia virus (MLV), human immunodeficiency virus (HIV), equineinfectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukaemia virus (Mo-MLV), FBR murine osteosarcorna virus (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus(A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV).

A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053-3058;Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

The vector of the present invention may be configured as a split-intronvector. A split intron vector is described in PCT patent applications WO99/15683 and WO 99/15684.

If the features of adenoviruses are combined with the genetic stabilityof retroviruses/lentiviruses then essentially the adenovirus can be usedto transduce target cells to become transient retroviral producer cellsthat could stably infect neighbouring cells. Such retroviral producercells engineered to express 5T4 antigen can be implanted in organismssuch as animals or humans for use in the treatment of angiogenesisand/or cancer.

The vector of the present invention may be configured as a psuedotypedvector.

In the design of retroviral vectors it may be desirable to engineerparticles with different target cell specificities to the native virus,to enable the delivery of genetic material to an expanded expanded oraltered range of cell types. One manner in which to achieve this is byengineering the virus envelope protein to alter its specificity. Anotherapproach is to introduce a heterologous envelope protein into the vectorparticle to replace or add to the native envelope protein of the virus.

The term pseudotyping means incorporating in at least a part of, orsubstituting a part of, or replacing all of, an env gene of a viralgenome with a heterologous env gene, for example an env gene fromanother virus. Pseudotyping is not a new phenomenon and examples may befound in WO 99/61639, WO-A-98/05759, WO-A-98/05754, WO-A-97/17457,WO-A-96/09400, WO-A-91/00047 and Mebatsion et al 1997 Cell 90, 841-847.

Pseudotyping can improve retroviral vector stability and transductionefficiency. A pseudotype of murine leukemia virus packaged withlymphocytic choriomeningitis virus (LCMV) has been described (Miletic etal (1999) J. Virol. 73:6114-6116) and shown to be stable duringultracentrifugation and capable of infecting several cell lines fromdifferent species.

Poxvirus Vectors

TAAs are weakly immunogenic, being recognised as “self” by the immunesystem and thus tolerated to a large extent. The use of poxvirus vectorsis sometimes able to cause the antigens to be presented such that thistolerance may be overcome at least in part, (especially if immuneevasion genes are deleted—see below) thus enabling a host to raise animmune response.

Poxvirus vectors are preferred for use in the present invention. Poxviruses are engineered for recombinant gene expression and for the useas recombinant live vaccines. This entails the use of recombinanttechniques to introduce nucleic acids encoding foreign antigens into thegenome of the pox virus. If the nucleic acid is integrated at a site inthe viral DNA which is non-essential for the life cycle of the virus, itis possible for the newly produced recombinant pox virus to beinfectious, that is to say to infect foreign cells and thus to expressthe integrated DNA sequence. The recombinant pox virus prepared in thisway can be used as live vaccines for the prophylaxis and/or treatment ofpathologic and infectious disease.

Expression of 5T4 peptide(s) in recombinant pox viruses, such asvaccinia viruses, requires the ligation of vaccinia promoters to thenucleic acid encoding the 5T4 peptide(s). Plasmid vectors (also calledinsertion vectors), have been constructed to insert nucleic acids intovaccinia virus through homologous recombination between the viralsequences flanking the nucleic acid in a donor plasmid and homologoussequence present in the parental virus (Mackett et al 1982 PNAS 79:7415-7419). One type of insertion vector is composed of: (a) a vacciniavirus promoter including the transcriptional initiation site; (b)several unique restriction endonuclease cloning sites located downstreamfrom the transcriptional start site for insertion of nucleic acid; (c)nonessential vaccinia virus sequences (such as the Thymidine Kinase (TK)gene) flanking the promoter and cloning sites which direct insertion ofthe nucleic acid into the homologous nonessential region of the virusgenome; and (d) a bacterial origin of replication and antibioticresistance marker for replication and selection in E. coli. Examples ofsuch vectors are described by Mackett (Mackett et al 1984, J. Virol. 49:857-864).

The isolated plasmid containing the nucleic acid to be inserted istransfected into a cell culture, e.g., chick embryo fibroblasts, alongwith the parental virus, e.g., poxvirus. Recombination betweenhomologous pox DNA in the plasmid and the viral genome respectivelyresults in a recombinant poxvirus modified by the presence of thepromoter-gene construct in its genome, at a site which does not affectvirus viability.

As noted above, the nucleic acid is inserted into a region (insertionregion) in the virus which does not affect virus viability of theresultant recombinant virus. Such regions can be readily identified in avirus by, for example, randomly testing segments of virus DNA forregions that allow recombinant formation without seriously affectingvirus viability of the recombinant. One region that can readily be usedand is present in many viruses is the thymidine kinase (TK) gene. Forexample, the TK gene has been found in all pox virus genomes examined[leporipoxvirus: Upton, et al J. Virology 60:920 (1986) (shope fibromavirus); capripoxvirus: Gershon, et al J. Gen. Virol. 70:525 (1989)(Kenya sheep-1); orthopoxvirus: Weir, et al J. Virol 46:530 (1983)(vaccinia); Esposito, et al Virology 135:561 (1984) (monkeypox andvariola virus); Hruby, et al PNAS, 80:3411 (1983) (vaccinia);Kilpatrick, et al Virology 143:399 (1985) (Yaba monkey tumour virus);avipoxvirus: Binns, et al J. Gen. Virol 69:1275 (1988) (fowlpox); Boyle,et al Virology 156:355 (1987) (fowlpox); Schnitzlein, et al J.Virological Method, 20:341 (1988) (fowlpox, quailpox); entomopox(Lytvyn, et al J. Gen. Virol 73:3235-3240 (1992)].

In vaccinia, in addition to the TK region, other insertion regionsinclude, for example, HindIII M.

In fowlpox, in addition to the TK region, other insertion regionsinclude, for example, BamHI J [Jenkins, et al AIDS Research and HumanRetroviruses 7:991-998 (1991)] the EcoRI-HindIII fragment, BamHIfragment, EcoRV-HindIII fragment, BamHI fragment and the HindIIIfragment set forth in EPO Application No. 0 308 220 A1. [Calvert, et alJ. of Virol 67:3069-3076 (1993); Taylor, et al Vaccine 6:497-503 (1988);Spehner, et al (1990) and Boursnell, et al J. of Gen. Virol 71:621-628(1990)].

In swinepox preferred insertion sites include the thymidine kinase generegion.

A promoter can readily be selected depending on the host and the targetcell type. For example in poxviruses, pox viral promoters should beused, such as the vaccinia 7.5K, or 40K or fowlpox C1. Artificialconstructs containing appropriate pox sequences can also be used.Enhancer elements can also be used in combination to increase the levelof expression. Furthermore, the use of inducible promoters, which arealso well known in the art, are preferred in some embodiments.

Foreign gene expression can be detected by enzymatic or immunologicalassays (for example, immuno-precipitation, radioimmunoassay, orimmunoblotting). Naturally occurring membrane glycoproteins producedfrom recombinant vaccinia infected cells are glycosylated and may betransported to the cell surface. High expressing levels can be obtainedby using strong promoters.

Other requirements for viral vectors for use in vaccines include goodimmunogenicity and safety. MVA is a replication-impaired vaccinia strainwith a good safety record. In most cell types and normal human tissue,MVA does not replicate. Replication of MVA is observed in a fewtransformed cell types such as BHK21 cells. Carroll et al (1997) haveshown that the recombinant MVA is equally as good as conventionalrecombinant vaccinia vectors at generating a protective CD8+T cellresponse and is an efficacious alternative to the more commonly usedreplication competent vaccinia virus. The vaccinia virus strains derivedfrom MVA, or independently developed strains having the features of MVAwhich make MVA particularly suitable for use in a vaccine, are alsosuitable for use in the present invention.

Preferably, the vector is a vaccinia virus vector such as MVA or NYVAC.Most preferred is the vaccinia strain modified virus ankara (MVA) or astrain derived therefrom. Alternatives to vaccinia vectors includeavipox vectors such as fowlpox or canarypox known as ALVAC and strainsderived therefrom which can infect and express recombinant proteins inhuman cells but are unable to replicate.

In one aspect of the present invention at least one immune evasion geneis deleted from the poxvirus vector.

Viruses, especially large viruses such a poxviruses which have anextensive coding capacity and can thus encode a variety of genes, havedeveloped a number of techniques for evading the immune system of theirhosts. For example, they are able to evade non-specific defences such ascomplement, interferons and the inflammatory response, as well as tointerfere with or block the function of cytokines. A number of theseimmune evasion polypeptides have been deleted from MVA, with theexception of the interferon resistance protein in the left terminalregion.

Poxviruses in general, being large DNA viruses which establish acute,rather than latent, infections. They encode so many antigenic proteinsthat antigenic variation is difficult, thus relying on active immuneevasion to protect themselves from the mammalian immune system. Theypossess a number of genes which encode polypeptides which areresponsible for interfering with a number of aspects of the immunesystem: they disrupt interferon action, interfere with complement,cytokine activity, inflammatory responses and CTL recognition (for areview, Smith et al., (1997) Immunol Rev 159:137-154). Removal of theseproteins is beneficial in promoting the ability of weak immunogensencoded on a poxvirus vector to elicit an immune response in a subject.

An immune evasion gene or polypeptide is a gene, or its product, whichassists the virus in evading the mammalian immune system. Preferably,the gene or gene product interferes with the working of the immunesystem, at least one level. This may be achieved in a number of ways,such as by interfering in signalling pathways by providing competitorsfor signalling molecules, by providing soluble cytokine receptor mimicsand the like.

Immune evasion genes include, but are not limited to, the following:

Interferon evasion genes. Vaccinia possesses at least three genes whichinterfere with IFN action. The E3L gene expresses a 25 Kd polypeptidewhich competes with P1 protein kinase for binding to dsRNA, an eventwhich leads to activation of P1, phosphorylation of eIF2α and resultantfailure of translation initiation complex assembly. This pathway isordinarily responsive to IFN activation, but is impeded by E3Lexpression thus allowing translation initiation to proceed unimpeded.

The K3L gene expresses a 10.5 Kd polypeptide which also interferes withP1 activity, since it is effectively an eIF2 mimic and acts as acompetitor for P1 protein kinase. Its mode of action is thus similar toE3L.

The A18R gene is predicted to encode a helicase, which appears tointerfere with the 2′,5′-oligoadenylate pathway, which is in turn IFNresponsive. 2′,5′-A activates RNAse L, which acts to prevent viraltranslation. Expression of A18R appears to reduce 2′,5′-A levels ininfected cells.

Complement. The product of the B5R gene of vaccinia is known to behighly related to factor H, a regulator of the alternative complementpathway. This pathway may be activated by antigen alone, unlike theclassical pathway. The B5R gene product thus may interfere with thealternative complement pathway.

The C21L gene is in turn related to C4b-binding protein in humans, andinteracts with cells bearing C4b on the surface to prevent binding tothe CR1 complement receptor.

Soluble Cytokine Receptors. The product of the vaccinia WR B15R gene(B16R in Copenhagen strain vaccinia) is related to IL1-R.

The WR gene ORF SalF19R, A53R in Copenhagen strain vaccinia, encodes aTNF receptor. However, in wild-type virus both of these genes arebelieved to be inactive due to fragmentation of the ORFs.

The B8R gene is believed to encode a soluble IFN-γ receptor, providingthe virus with yet another IFN evasion mechanism.

Inflammation. A number of genes are believed to be involved in theprevention of inflammatory responses to viral infection. These includeA44L, K2L, B13R and B22R.

In one aspect of the present invention, the majority of the immuneevasion genes are deleted from the recombinant poxvirus vector.Preferably, all the immune evasion genes are deleted. Thus, in oneaspect of the present invention, the recombinant poxvirus vector is arecombinant MVA vector in which the K3L interferon resistance proteingene has been disrupted or deleted.

Preferred are poxviruses which are non-hazardous to the intendedsubject. Thus, for example, for use in humans, poxviruses which areeither host-range restricted, such as avipox viruses, or otherwiseattenuated, such as attenuated strains of vaccinia (including NYVAC andMVA) are preferred. Most preferred are attenuated vaccinia virusstrains, although non-vaccinia strains are usefully employed in subjectswith pre-existing smallpox immunity.

A construct which contains at least one nucleic acid which codes for 5T4epitope(s) flanked by MVA DNA sequences adjacent to a naturallyoccurring deletion, e.g. deletion II, within the MVA genome, isintroduced into cells infected with MVA, to allow homologousrecombination.

Once the construct has been introduced into the eukaryotic cell and the5T4 epitope DNA has recombined with the viral DNA, the desiredrecombinant vaccinia virus, can be isolated, preferably with the aid ofa marker (Nakano et al Proc. Natl. Acad. Sci. USA 79, 1593-1596 [1982],Franke et al Mol. Cell. Biol. 1918-1924 [1985], Chakrabarti et al Mol.Cell. Biol. 3403-3409 [1985], Fathi et al Virology 97-105 [1986]).

The construct to be inserted can be linear or circular. A circular DNAis preferred, especially a plasmid. The construct contains sequencesflanking the left and the right side of a naturally occurring deletion,e.g. deletion II, within the MVA genome (Altenburger, W., Suter, C. P.and Altenburger J. (1989) Arch. Virol. 105, 15-27). The foreign DNAsequence is inserted between the sequences flanking the naturallyoccurring deletion.

For the expression of at least one nucleic acid, it is necessary forregulatory sequences, which are required for the transcription of thenucleic acid to be present upstream of the nucleic acid. Such regulatorysequences are known to those skilled in the art, and includes forexample those of the vaccinia 11 kDa gene as are described inEP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385).

The construct can be introduced into the MVA infected cells bytransfection, for example by means of calcium phosphate precipitation(Graham et al Virol. 52, 456-467 [1973; Wigler et al Cell 777-785 [1979]by means of electroporation (Neumann et al EMBO J. 1, 841-845 [1982]),by microinjection (Graessmann et al Meth. Enzymology 101, 482492(1983)), by means of liposomes (Straubinger et al Methods in Enzymology101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl.Acad. Sci. USA 77, 2163-2167 (1980)) or by other methods known to thoseskilled in the art. Transfection by means of liposomes is preferred.

The recombinant priming and boosting vectors of the present inventioncan have a tropism for a specific cell type in the mammal. By way ofexample, the recombinant vectors of the present invention can beengineered to infect professional APCs such as dendritic cells andmacrophages. Dendritic cells are known to be orchestrators of asuccessful immune response especially that of a cell mediated response.It has been shown that ex vivo treatment of dendritic cells with antigenor viral vectors containing such a target antigen, will induceefficacious immune responses when infused into syngeneic animals orhumans (see Nestle F O, et al. Vaccination_of melanoma patients withpeptide- or tumor lysate-pulsed dendritic cells, Nat Med. 1998 March;4(3):328-32 and Kim C J, et al. Dendritic cells infected with poxvirusesencoding MART-1/Melan A sensitize T lymphocytes in vitro. J Immunother.1997 July; 20(4):276-86. The recombinant vectors can also infect tumourcells. Alternatively, the recombinant vectors are able to infect anycell in the mammal.

Other examples of vectors include ex vivo delivery systems, whichinclude but are not limited to DNA transfection methods such aselectroporation, DNA biolistics, lipid-mediated transfection andcompacted DNA-mediated transfection.

The vector may be a plasmid DNA vector. As used herein, “plasmid” refersto discrete elements that are used to introduce heterologous DNA intocells for either expression or replication thereof. Selection and use ofsuch vehicles are well within the skill of the artisan.

Pulsed Cells

The present invention also provides cells pulsed with peptides of thefirst aspect of the invention.

Preferably the cells to be pulsed are capable of expressing MHC class Ior class II.

MHC class I molecules can be expressed on nearly all cell types, butexpression of MHC class II molecules is limited to so-called“professional” antigen presenting cells (APCs); B cells, dendritic cellsand macrophages. However, expression of MHC class II can be induced onother cell types by treating with IFNγ.

Expression of MHC class I or MHC class II molecules can also be achievedby genetic engineering (i.e. provision of a gene encoding the relevantMHC molecule to the cell to be pulsed). This approach has the advantagethat an appropriate MHC haplotype(s) can be chosen which bindspecifically to the peptide(s).

Preferably the cell to be pulsed is an antigen presenting cell, i.e. acell which, in a normal immune response, is capable of processing anantigen and presenting it at the cell surface in conjunction with an MHCmolecule. Antigen presenting cells include B cells, macrophages anddendritic cells. In an especially preferred embodiment, the cell is adendritic cell.

Preferably the cell is capable of expressing an MHC molecule which bindsa peptide according to the first aspect of the invention in its peptidebinding groove. For example, the cell may express one of the followingHLA restriction elements: B8, Cw7 or A2 (for MHC class I).

Peptide pulsing protocols are known in the art (see for exampleRedchenko and Rickinson (1999) J. Virol. 334-342; Nestle et al (1998)Nat. Med. 4 328-332; Tjandrawan et al (1998) J. Immunotherapy 21149-157). For example, in a standard protocol for loading dendriticcells with peptides, cells are incubated with peptide at 50 μg/ml with 3μg/ml β-2 microglobulin for two hours in serum free medium. The unboundpeptide is then washed off.

The pulsed cell of the present invention may be used as a vaccine, forexample to stimulate a prophylactic or therapeutic anti-5T4 immuneresponse.

The present invention therefore also provides a method for treatingand/or preventing a disease which comprises the step of administering apeptide-pulsed cell to a subject in need of same.

Vaccine/Pharmaceutical Composition

The present invention also provides a vaccine/pharmaceutical compositioncomprising a peptide epitope, a polyepitope string, a nucleic acidsequence, a vector system and/or a cell according to previous aspects ofthe invention.

The vaccine/pharmaceutical composition may be for prophylactic ortherapeutic use.

The vaccine may by prepared as an injectable, either as liquid solutionor suspension; solid form suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified, or the protein encapsulated in liposomes. The activeimmunogenic ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof.

In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

Further examples of adjuvants and other agents include aluminiumhydroxide, aluminium phosphate, aluminium potassium sulphate (alum),beryllium sulphate, silica, kaolin, carbon, water-in-oil emulsions,oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers orother synthetic adjuvants. Such adjuvants are available commerciallyfrom various sources, for example, Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.).

Typically, adjuvants such as AMPHIGEN™ (oil-in-water), ALHYDROGEL™(aluminium hydroxide), or a mixture of AMPHIGEN™ and ALHYDROGEL™adjuvants are used. Only aluminium hydroxide is approved for human use.

The proportion of immunogen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. For example,aluminium hydroxide can be present in an amount of about 0.5% of thevaccine mixture (Al₂O₃ basis). Conveniently, the vaccines are formulatedto contain a final concentration of immunogen in the range of from 0.2to 200 μg/ml, preferably 5 to 50 μg/ml, most preferably 15 μg/ml.

After formulation, the vaccine may be incorporated into a sterilecontainer which is then sealed and stored at a low temperature, forexample 4° C., or it may be freeze-dried. Lyophilisation permitslong-term storage in a stabilised form.

The vaccine may be administered in a convenient manner such as by theoral, intravenous (where water soluble), intramuscular, subcutaneous,intranasal, intradermal or suppository routes or implanting (e.g. usingslow release molecules).

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1% to 2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10% to 95% of activeingredient, preferably 25% to 70%. Where the vaccine composition islyophilised, the lyophilised material may be reconstituted prior toadministration, e.g. as a suspension. Reconstitution is preferablyeffected in buffer.

Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, EUDRAGIT™coating “S”, EUDRAGIT™ coating “L”, cellulose acetate, cellulose acetatephthalate or hydroxypropylmethyl cellulose.

5T4 peptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids such as acetic, oxalic, tartaric and maleic. Saltsformed with the free carboxyl groups may also be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine and procaine.

Heterologous Vaccination Regimes

Regimes for administration of vaccines/pharmaceutic compositionsaccording to the present invention may be determined by conventionalefficacy testing. Especially preferred, however, are regimes whichinclude successive priming and boosting steps. It is observed that suchregimes achieve superior breaking of immune tolerance and induction of Tcell responses (see Schneider et al., 1998 Nat Med 4:397-402).

Prime-boost regimes may be homologous (where the same composition isadministered in subsequent doses) or heterologous (where the primimg andboosting compositions are different). For example, the primingcomposition may be a non-viral vector (such as a plasmid) encoding a 5T4antigen and the boosting composition may be a viral vector (such as apoxvirus vector) encoding a 5T4 antigen, wherein either or both of said“5T4 antigens” is an epitope or polyepitope string of the presentinvention.

Diagnostic Methods

The present invention also provides an agent capable of bindingspecifically to a peptide according to the present invention and/or anucleic acid sequence which encodes such a peptide.

An agent is considered to “bind specifically” to a peptide/nucleic acidsequence of the present invention if there is a greater than 10 folddifference, and preferably a 25, 50 or 100 fold difference between thebinding of the agent to a peptide/nucleic acid sequence of the presentinvention and another peptide/nucleic acid sequence.

The agent may be any compound capable of binding specifically to apeptide and/or a nucleic acid sequence. The term “compound” refers to achemical compound (naturally occurring or synthesised), such as abiological macromolecule (e.g., nucleic acid, protein, non-peptide, ororganic molecule), or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian) cells ortissues, or even an inorganic element or molecule.

Preferably the agent is identifiable by screening a library of candidatecompounds. Libraries of compounds may be screened in multi-well plates(e.g., 96-well plates), with a different test compound in each well. Inparticular, the library of candidate compounds may be a combinatoriallibraries. A variety of combinatorial libraries of random-sequenceoligonucleotides, polypeptides, or synthetic oligomers have beenproposed and number of small-molecule libraries have also beendeveloped. Combinatorial libraries of oligomers may be formed by avariety of solution-phase or solid-phase methods in which mixtures ofdifferent subunits are added stepwise to growing oligomers or parentcompound, until a desired oligomer size is reached (typicallyhexapeptide or heptapeptide). A library of increasing complexity can beformed in this manner, for example, by pooling multiple choices ofreagents with each additional subunit step. Alternatively, the librarymay be formed by solid-phase synthetic methods in which beads containingdifferent-sequence oligomers that form the library are alternately mixedand separated, with one of a selected number of subunits being added toeach group of separated beads at each step. Libraries, includingcombinatorial libraries are commercially available from pharmaceuticalcompanies and speciality library suppliers.

Where the agent recognises a nucleic acid according to the presentinvention, the agent may comprise an antisense sequence.

Where the agent recognises a peptide according to the present invention,the agent may comprise an MHC molecule or part thereof which comprisesthe peptide binding groove. Alternatively the agent may comprise ananti-peptide antibody.

As used herein, “antibody” includes a whole immunoglobulin molecule or apart thereof or a bioisostere or a mimetic thereof or a derivativethereof or a combination thereof. Examples of a part thereof include:Fab, F(ab)′₂, and Fv. Examples of a bioisostere include single chain Fv(ScFv) fragments, chimeric antibodies, bifunctional antibodies.

The term “mimetic” relates to any chemical which may be a peptide,polypeptide, antibody or other organic chemical which has the samebinding specificity as the antibody.

The term “derivative” as used herein in relation to antibodies includeschemical modification of an antibody. Illustrative of such modificationswould be replacement of hydrogen by an alkyl, acyl, or amino group.

A whole immunoglobulin molecule is divided into two regions: binding(Fab) domains that interact with the antigen and effector (Fc) domainsthat signal the initiation of processes such as phagocytosis. Eachantibody molecule consists of two classes of polypeptide chains, light(L) chains and heavy (H) chains. A single antibody has two identicalcopies of the L chain and two of the H chain. The N-terminal domain fromeach chain forms the variable regions, which constitute theantigen-binding sites. The C-terminal domain is called the constantregion. The variable domains of the H (V_(H)) and L (V_(L)) chainsconstitute an Fv unit and can interact closely to form a single chain Fv(ScFv) unit. In most H chains, a hinge region is found. This hingeregion is flexible and allows the Fab binding regions to move freelyrelative to the rest of the molecule. The hinge region is also the placeon the molecule most susceptible to the action of protease which cansplit the antibody into the antigen binding site (Fab) and the effector(Fc) region.

The domain structure of the antibody molecule is favourable to proteinengineering, facilitating the exchange between molecules of functionaldomains carrying antigen-binding activities (Fabs and Fvs) or effectorfunctions (Fc). The structure of the antibody also makes it easy toproduce antibodies with an antigen recognition capacity joined tomolecules such as toxins, lymphocytes or growth factors.

Chimeric antibody technology involves the transplantation of whole mouseantibody variable domains onto human antibody constant domains. Chimericantibodies are less immunogenic than mouse antibodies but they retaintheir antibody specificity and show reduced HAMA responses.

In chimeric antibodies, the variable region remains completely murine.However, the structure of the antibody makes it possible to producevariable regions of comparable specificity which are predominantly humanin origin. The antigen-combining site of an antibody is formed from thesix complementarity-determining regions (CDRs) of the variable portionof the heavy and light chains. Each antibody domain consists of sevenantiparallel β-sheets forming a β-barrel with loops connecting theβ-strands. Among the loops are the CDR regions. It is feasible to morethe CDRs and their associated specificity from one scaffolding β-barrelto another. This is called CDR-grafting. CDR-grafted antibodies appearin early clinincal studies not to be as strongly immunogenic as eithermouse or chimaeric antibodies. Moreover, mutations may be made outsidethe CDR in order to increase the binding activity thereof, as inso-called humanised antibodies.

Fab, Fv, and single chain Fv (ScFv) fragments with VH and VL joined by apolypeptide linker exhibit specificities and affinities for antigensimilar to the original monoclonal antibodies. The ScFv fusion proteinscan be produced with a non-antibody molecule attached to either theamino or carboxy terminus. In these molecules, the Fv can be used forspecific targeting of the attached molecule to a cell expressing theappropriate antigen. Bifunctional antibodies can also be created byengineering two different binding specificities into a single antibodychain. Bifunctional Fab, Fv and ScFv antibodies may comprise engineereddomains such as CDR grafted or humanised domains.

Procedures for identifying, characterising, cloning and engineeringpolyclonal and monoclonal antibodies and their derivatives are wellestablished, for example using hybridomas derived from mice ortransgenic mice, phage-display libraries or scFv libraries. Genesencoding immunoglobulins or immunoglobulin-like molecules can beexpressed in a variety of heterologous expression systems. Largeglycosylated proteins including immunoglobulins are efficiently secretedand assembled from eukaryotic cells, particularly mammalian cells.Small, non-glycosylated fragments such as Fab, Fv, or scFv fragments canbe produced in functional form in mammalian cells or bacterial cells.

The agent may recognise the peptide/nucleic acid of the presentinvention alone, or in conjunction with another compound. For example,the agent may be capable or binding specifically to the peptide whenpresented by an MHC molecule. In this case, the agent of the presentinvention may comprise a T cell receptor molecule or part thereof.

The T cell receptor may be associated with another molecule such as CD4(for MHC class II epitopes) or CD8 (for MHC class I epitopes).Alternatively, or in addition, the receptor may be associated with CD3.

If the agent occurs naturally in the human body, then preferably theagent of the present invention is in a substantially isolated form.

The present invention also provides a method which comprises the step ofdetecting the presence of a peptide, nucleic acid or agent of thepresent invention in a subject.

In a preferred embodiment, the method is used to detect the presence ofT cells capable of specifically recognising a peptide epitope accordingto the present invention in conjunction with an MHC molecule.

The diagnostic method may, for example, be for diagnosing or monitoringthe progression of a disease or for monitoring the progression of animmune response in a subject.

As mentioned above, as an immune response progresses, the dominance ofparticular epitopes may change, and sub-dominant epitopes canpredominate. Thus by detecting the presence of a particular epitope, ora TCR/T cell capable of recognising such an epitope, information can begained about the progression of the immune response.

The method may be carried out in vivo, or more preferably on an ex vivosample.

Thus the present invention also provides a diagnostic method whichcomprises the following steps:

-   -   (i) isolating a sample from a subject;    -   (ii) detecting in the sample ex vivo the presence of T cells        capable of specifically recognising a peptide epitope according        to the present invention in conjunction with an MHC molecule.

In a preferred embodiment, the method is for diagnosing or monitoringthe progression of a cancerous disease.

The nature of the method will depend on whether a peptide, nucleic acidor agent of the present invention is being detected (and if it is anagent, on the nature of that agent).

In order to detect a peptide of the present invention, an agent of thepresent invention (such as an antibody or an MHC molecule) may be used.Methods of screening with antibodies (such as ELISAs, immunoblotting,western blotting, competitive assays, two site capture assays) are wellknown in the art.

In order to detect peptides or specific T cells, an antigen presentationassay may be used. When a T cell successfully recognises an MHC:peptidecomplex, it is stimulated. This stimulation can be monitored byproliferation of the T cells (for example by incorporation of ³H) and/orby production of cytokines by the T cells (for example by an ELISPOTassay). Thus it is possible to detect the presence of a specific peptideby using appropriate APCs and T cells lines, and to detect the presenceof a specific T cell by using appropriate APCs and peptide/antigen.

The presence of a particular cell surface molecule (such as a TCR or MHCmolecule) can also be investigated using fluorescence activated cellscanning (FACS).

Where the method is to detect the presence of a nucleic acid, numerousmethods are known in the art such as PCR, southern blotting (for DNA)and northern blotting (for RNA).

T Cells

The present invention also relates to a T cell, such as a T cell clone,or line, which is capable of specifically recognising a peptide epitopeaccording to the present invention in conjunction with an MHC molecule.Several methods for generating T cell lines and clones are known in theart. One method for generating T cell lines is as follows:

Mice are primed with antigen (usually subcutaneously in the rearfootpad), and the draining lymph nodes (in this case the popliteal andinguinal) are removed 1 week later and set up in co-culture with theantigen and with syngeneic feeder cells i.e. cells from mice of the sameinbred line (e.g. normal thymocytes or splenocytes). After 4 days thelymphoblasts are isolated and induced to proliferate with IL-2. When thepopulation of cells has expanded sufficiently, they are checked forantigen and MHC specificity in a lymphocyte transformation test, and aremaintained by alternate cycles of culture on antigen-treated feedercells and culture in IL-2-containing medium.

The definitive T-cell lineage marker is the T-cell receptor (TCR). Thereare presently two defined types of TCR, both of which are heterodimersof two disulphide-linked polypeptides. One type consist of α and βchains, the other type consists of γ and δ chains. Approximately 90-95%of blood T cells express α/β TCR, the other 5-10% expressing γ/δ TCR.

T cells can be divided into two distinct populations: a subset whichcarries the CD4 marker and mainly “helps” or “induces” immune responses(T_(H)) and a subset which carries the CD8 marker and is predominantlycytotoxic (T_(C)). CD4+ T cells recognise peptides in association withMHC class II molecules, whereas CD8+ T cells recognise peptides inassociation with Class I molecules, so the presence of CD4 or CD8restricts the types of cell with which the T cell can interact.

The CD4 set has been functionally sub-divided into two further subsets:

-   (i) T cells that positively influence the response of T cells and B    cells (the helper T cell function) are CD29+. Practically all the    cells in this population also express a low molecular weight isoform    of the CD45 leucocyte common antigen, designated CD45RO.-   (ii) Cells that induce the supressor/cytotoxic functions of CD8+    cells (the suppressor/inducer function) express a different form of    the CD45 molecule, CD45RA.

Functional diversity has also been demonstrated by functional analysisof T_(H) clones for cytokine secretion patterns. The T_(H)1 subset ofCD4+ T cells secrete IL-2 and IFN-γ, whereas the T_(H)2 subset producesIL-4, L-5, IL-6 and IL-10. T_(H)1 cells mediate several functionsassociated with cytotoxicity and local inflammatory reactions.Consequently these cells are important for combatting intracellularpathogens, including viruses, bacteria and parasites. T_(H)2 cells aremore effective at stimulating B cells to proliferate and produceantibodies, and therefore in normal immune responses function to protectagainst free-living organisms.

Expression of all of the markers described above can readily be detectedusing specific antibodies, so the type of T cell can beselected/determined using FACS. Expression of particular cytokines canalso be detected by methods known in the art, such as ELISPOT assay.

Prophylactic/Therapeutic Methods

The present invention also provides the use of a vaccine according tothe present invention in the manufacture of a medicament for use in theprevention and/or treatment of a disease.

There is also provided a method for treating and/or preventing a diseasein a subject which comprises the step of administering an effectiveamount of a vaccine according to the present invention.

Administration of the vaccine may elicit an immune response in thesubject. In a preferred embodiment, administration of the vaccine breaksimmune tolerance to 5T4 in the subject.

Where the peptide is a class I epitope, the immune response elicited mayinvolve the activation of 5T4 specific cytotoxic T-lymphocytes. Wherethe peptide is a class II epitope, the immune response elicited mayinvolve the activation of T_(H)1 and/or T_(H)2 cells.

Advantageously, the response is an anti-tumour immunotherapeuticresponse which is effective to inhibit, arrest or reverse thedevelopment of a tumour in a subject.

Combination Therapies

The invention further relates to the use of 5T4 targeting molecules,such as anti-5T4 antibodies, for example anti-5T4 scFvs. Theseantibodies may be used to (i) to target natural or exogenous 5T4 in situand/or (ii) deliver immune enhancer molecules, such as B7.1, to naturalor exogenous 5T4 in situ (Carroll et al. (1998) J Natl Cancer Inst90(24):1881-7). This potentiates the immunogenicity of 5T4 in thesubject.

The present invention thus also relates to the sequential use of avaccine according to the present invention and anti-5T4 antibodies, forexample anti-5T4 scFvs. The anti-5T4 scFvs antibodies may beadministered as naked DNA encoding the antibodies (for example, in aplasmid comprising the encoding DNA together with a short promoterregion to control its production), in an expression vector (which may beviral or non-viral) comprising the encoding sequence or in a proteinform. Thus, the invention provides a vector encoding a 5T4 peptideantigen and an agent capable of binding 5T4 which is optionally fusedwith an immunostimulatory molecule, for separate, such as sequentialuse, in the treatment of tumours.

In a further embodiment, the invention encompasses a combination therapyincluding enzyme/prodrug therapy and immunotherapy with 5T4. Forexample, the enzyme/prodrug therapy may comprise intratumoural orsystemic delivery of P450, delivered optionally using an retroviral orlentiviral vector, and cyclophosphamide (CPA) followed by systemicimmunotherapeutic induction with 5T4.

Thus, the invention further relates to a vector encoding 5T4 peptideantigen and a prodrug/enzyme combination, for separate, simultaneousseparate or combined use in the treatment of tumours.

Diseases

5T4 is a tumour associated antigen. Presence of 5T4 on cancer cells isassociated with metastasis and has been shown to be an independentindicator of prognosis in a number of different cancers.

In a preferred embodiment, the disease (which is preventable/treatableusing a vaccine according to the present invention) is a cancer. Inparticular the disease may be a carcinoma of, for example, the breast,lung, stomach, pancreas, endometrium, cervix, colorectal, renal orprostate.

WO89/07947 describes an immunohistochemical screen of neoplastic tissuesusing an anti-5T4 monoclonal antibody (see Tables II and VI).Preferably, the disease is a cancer which can be shown to be 5T4positive by diagnostic testing (such as with an anti-5T4 antibody), forexample: an invasive carcinoma of the Ampulla of Vater, breast, colon,endometrium, pancreas, or stomach; a squamous carcinoma of the bladder,cervix, lung or oesophagus; a tubulovillous adenoma of the colon; amalignant mixed Mullerian tumour of the endometirem; a clear cellcarcinoma of the kidney; a lung cancer (large cell undifferentiated,giant cell carcinoma, broncho-alveolar carcinoma, metastaticleiomyosarcoma); an ovarian cancer (a Brenner tumour,cystadenocarcinoma, solid teratoma); a cancer of the testis (seminoma,mature cystic teratoma); a soft tissue fibrosarcoma; a teratoma(anaplastic germ cell tumours); or a trophoblast cancer (choriocarcimoma(e.g. in uterus, lung or brain), tumour of placental site, hydatidiformmole).

Tetramers

The present invention also provides 5T4 peptide epitope associated with(eg. folded with) tetramers and uses thereof.

Tetramers are fluorescent reagents that allow for the directvisualisation of antigen-specific T-cells (Altman et al. (1996) Science271, 94-96). They consist of individual peptides epitopes refolded withHLA class I protein and bind to T cells that are specific for thatparticular epitope. They allow for the direct quantification of antigenspecific lymphocytes and have been applied widely in human and murineimmunology.

The tetramers may be prepared using the methods described by Altman etal. (1996) Science 271, 94-96. Briefly, tetramers may be prepared byadding biotinylated protein to streptavidin PE at a ratio of 4:1.Tetramer bound cells may be selected using magnetic activated cellsorting (MACS). MACS has been described in Radbruch et al. (1994)Methods in Cell Biology 42, 387-403.

Advantageously, the use of tetramers allows for the tracking of a5T4-specific immune response before, during and after vaccination; topurify autologous CD4+ T cells from individual patients andexpand/manipulate them ex vivo for possible re-infusion; as a diagnosticindicator, for example, in subjects prone to colorectal and other5T4-positive cancers. Accordingly, the present invention also relates tothe use of a 5T4 peptide epitope tetramer for monitoring a 5T4-specificimmune response before, during or after vaccination. The presentinvention further relates to the use of a 5T4 peptide epitope tetramerfor the purification of autologous CD4+ T cells from individualpatients. The present invention still further relates to the use of a5T4 peptide epitope tetramer as a diagnostic indicator in subjects proneto 5T4-positive cancers—such as colorectal cancers.

The invention is further described, for the purposes of illustrationonly, in the following examples in which reference is made to thefollowing Figures.

FIG. 1 is a schematic diagram illustrating the proliferation assay formeasuring cellular responses

FIG. 2 is a schematic diagram illustrating the ELISPOT assay formeasuring antigen-specific IFNγ production.

FIG. 3 shows the proliferative responses of breast cancer patientsOBCR24 and OBCR25 following stimulation in vitro with h5T4 peptide pools

FIG. 4 shows proliferative responses of PBMCs from OBCR25 followingstimulation in vitro with h5T4 peptides

FIG. 5 shows an ELISPOT assay to show IFN-γ secretion by PBMCs fromOBCR24 and OBCR25 following stimulation in vitro with antigens

FIG. 6 shows an ELISPOT assay to show IFN-γ secretion by PBMCs fromOBCR25 following stimulation in vitro with single h5T4 peptides

FIG. 7 shows proliferative responses of PBMCs from OBCR27 followingstimulation in vitro with h5T4 peptide pools.

FIG. 8 shows an ELISPOT assay to show IFN-γ secretion by PBMCs fromOBCR27 following stimulation in vitro with h5T4 peptide pools.

FIG. 9 shows proliferative responses of PBMCs from TV1-104 at 2 and 4weeks post-vaccination following stimulation in vitro with h5T4 peptides

FIG. 10 shows an ELISPOT assay to show IFN-γ secretion by PBMCs fromTV1-104 at 2 and 4 weeks post-vaccination following stimulation in vitrowith antigens

EXAMPLES Example 1 Non TROVAX® Patients Example 1A Breast CancerPatients OBCR25

A h5T4 peptide library was produced consisting of 51 20 mer peptidesoverlapping by 8 amino acids. Class II peptide pools containing 5peptides per pool were incubated with PBMCs from breast cancer patientsOBCR24 and OBCR25 and proliferation was measures using the assayillustrated in FIG. 1. After 6 days incubation, cells were pulsedovernight with ³H-thymidine and incubated overnight at 37° C. It wasfound that peptides present in pool #10 elicit a strong proliferativeresponse with OBCR25 cells (FIG. 3).

Dissection of the proliferative response seen to peptide pool 10revealed that the positive response resides in the 20 mer peptide #51(FIG. 4).

To measure antigen-specific IFNγ production, an ELISPOT assay wasconducted following the general scheme illustrated in FIG. 2. IFNγELISPOT of PBMCs from OBCR25 following restimulation in vitro with h5T4peptides shows that the positive ELISPOT response observed in pool #10resides entirely in peptide #51 (FIG. 5).

The “pepset” peptide comprise the overlapping peptides spanning theentire sequence of 5T4. As they are mass produced, the level of purityis not very high. To rule out the possibility of contamination, thepresent inventors repeated the experiment using peptide 51 resynthesisedto high purity (FIG. 6).

Peptide ˜51 comprises the extreme C-terminal 20 amino acids of h5T4:

-   -   ⁴⁰¹YRYEINADPRLTNLSSNSDV⁴²⁰ (SEQ ID NO:2)

This sequence shows no significant homology to any other proteinsequence deposited in accessible databases.

By using truncated versions of peptide #51, it is possible to determinethe minimal epitope of this peptide.

HLA Typing of OBCR25

PBMCs from patient OBCR25 were HLA typed, giving the following results:

OBCR25: HLA-A *2402/5/9/11/14-15/ *2601/11-12/14-15 20/25-27/30 HLA-B*5001/4 *5101/3/9/11/14/16-18/21 HLA-Cw *0602-3/7 *1502-6/9 HLA-DRB1*0701 *1301/28/35 HLA-DRB3 *0201-3/5/11/13 HLA-DRB4 *0101-5 HLA-DQB1*0201-2 *0602-4/7-8/10-11/13-16

Example 1B Duke's B Colon Cancer Patient OBCR27

PBMCs from OBCR27 were stimulated in vitro with h5T4 class II peptidepools and proliferative responses were measured using the assay outlinedin FIG. 1. Strong proliferative responses were seen to peptide pools 8,9 and 10 (FIG. 7).

An IFNγ ELISPOT assay (as outlined in FIG. 2) was also conducted usingclass II peptide pools. Positive ELISPOT responses were seen to peptidepools 8, 9 and 10 (FIG. 8).

By analysing the individual peptides from each pool, it is possible toestablish which one(s) are responsible for the observed positiveproliferative and ELISPOT responses, and which therefore are or comprisean h5T4 class II epitope.

Example 2 TROVAX® Colorectal Patients

Throughout the phase I/II TROVAX® clinical trial, patients weremonitored for their ability to mount an immune response to the h5T4overlapping peptide library.

Of 8 people recruited to the trial, 7 have shown a response to one ormore peptides post-boost, but not prior to vaccination.

Looking at one patient in particular (TV1-104) proliferation of PBMCsfrom this patient to different antigen preparations were examined at 2and 4 weeks post-vaccination, using the assay outlined in FIG. 1. Potentproliferative responses are observed to peptides present in pool #9 and,in particular its constituent peptide 45 (FIG. 9). A potent IFNγ ELISPOTresponse was also induced by peptide present in pool #9 and inparticular its constituent peptide #45 (FIG. 10) using the assay shownin FIG. 2.

By using truncated versions of peptide #45, it is possible to determinethe minimal epitope of this peptide.

HLA Typing of TV1-104

PBMCs from patient TV1-104 were HLA typed, giving the following results:

TV1-104: HLA-A *0206-7/10/14-15/17-18/ *0301-2 21/33/39/41 HLA-B*0702/4/15/22-23/25 *4001/22/30/32/34 HLA-Cw *0702/13/15 *0304-6/8-9/14HLA-DRB1 *0404/8/19/22-23/25/ *1301/28/35 31-32/36/42 HLA-DRB3 *0101/3-6HLA-DRB4 *0101-5 HLA-DQB1 *0302/7-8 *0602-4/7-8/10-11/13-16

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1. An isolated peptide consisting of the amino acid sequence of SEQ IDNO:
 2. 2. A polyepitope string comprising two or more peptides, whereineach of the peptides consists of the of SEQ ID NO:
 2. 3. A compositioncomprising an isolated peptide according to claim 1 in association witha cell penetrator.
 4. A composition comprising a polyepitope stringaccording to claim 2 in association with a cell penetrator.
 5. Acomposition comprising a peptide consisting of the amino acid sequenceof SEQ ID NO: 2 bound to an adjuvant.